Antiviral Therapy 6:

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1 Antiviral Therapy 6: Low-rate emergence of thymidine analogue mutations and multi-drug resistance mutations in the HIV-1 reverse transcriptase gene in therapynaive patients receiving stavudine plus lamivudine combination therapy Mireille Mouroux 1, Diane Descamps 2, Jacques Izopet 3, Anne Yvon 1, Constance Delaugerre 1, Sophie Matheron 2, Anne Coutellier 1, Marc-Antoine Valantin 1, Manuella Bonmarchand 1, Henri Agut 1, Patrice Massip 3, Dominique Costagliola 4, Christine Katlama 1, Françoise Brun-Vezinet 2 and Vincent Calvez 1 * 1 Departments of Virology, Infectious Diseases and Internal Medecine, Pitié-Salpêtrière Hospital, Paris, France 2 Departments of Virology and Infectious Diseases, Bichat Claude Bernard Hospital, Paris, France 3 Departments of Virology and Infectious Diseases, Purpan Hospital, Toulouse, France 4 INSERM SC4, Saint Antoine Hospital, Paris, France *Corresponding author: Tel: ; Fax: ; vincent.calvez@psl.ap-hop-paris.fr. Objectives: Mutations usually associated with zidovudine exposure have been observed in zidovudine-naive patients treated by stavudine in combination. These mutations were named thymidine analogue mutations (TAMs). This fact, combined with phenotypical and biochemical findings provided additional evidence for cross-resistance between zidovudine and stavudine. A recent genotypic study in naive patients receiving stavudine/didanosine combination showed emergence of TAMs and a multidrug-resistance mutation (MDR), Q151M, in 36 and 10% of cases, respectively. Stavudine plus lamivudine is one of the most used binucleoside associations in the antiretroviral combinations. The objective of this study was to assess the genotypic changes in the HIV-1 reverse transcriptase (RT) gene in antiretroviralnaive patients treated by stavudine plus lamivudine. Methods: We analysed the RT gene of 44 HIV-1 patients, naive of antiretroviral therapy, who were treated for 24 or 48 weeks with stavudine/lamivudine. Results: At the end of the follow-up, all patients acquired the lamivudine-associated mutation M184V. Only two subjects (4.5%) developed a TAM (T215Y; M41L), one subject developed a V75T/A mutation and one subject developed the particular MDR pattern F116Y, Q151M. Conclusions: Our study clearly demonstrated that naive subjects treated with stavudine/lamivudine for weeks selected a low rate of TAMs and MDR Q151M. One hypothesis explaining these results could be the development of the M184V mutation. Introduction Stavudine (2,3,-didehydro-3 deoxythymidine) is a potent inhibitor of HIV-1 reverse transcriptase (RT) in vitro and in vivo. This drug has been shown in several clinical trials to be particularly effective when used in combination with another nucleoside analogue and a protease inhibitor. Among the approved antiretrovirals, stavudine has been the most difficult to demonstrate specific phenotypic and genotypic resistance in the clinic, despite prolonged treatment periods [1,2]. Stavudine based antiretroviral combination is less effective in zidovudine-experienced than in treatment-naive subjects [3]. A stavudine resistance mutation (V75T) in the HIV-1 RT has been reported following in vitro passages, but its effects seem to be virus strain-related. The V75T change appears very infrequently in clinical isolates from stavudine-treated patients, and no apparent change in stavudine sensibility has been noted. Isolates reported to have reduced sensitivity to stavudine also display resistance to several other nucleoside inhibitors. These nucleoside multi-resistant variants emerge through several mechanisms, involving either selection of the Q151M multidrugresistance pattern or insertions of several amino acids (mainly serine) at codon 69. These multi-resistant patterns appeared in patients who have been on 2001 International Medical Press /01/$

2 M Mouroux et al. sequential or combined nucleoside analogue therapies for prolonged periods. Stavudine resistance might also be achieved through changes that appear to map outside the RT gene [1,2]. Recently, a new concept of mutations was born, based on the fact that chemical structures of stavudine and zidovudine are very similar (thymidine analogues), and differ only on the presence of a radical (N3). These mutations have been described to be associated with use of zidovudine and stavudine, and were named thymidine analogue mutations (TAM). More recently, biochemical findings provided additional evidence of cross resistance between zidovudine and stavudine. Purified HIV-1 RT from zidovudine-resitant isolates demonstrated reduced inhibition by zidovudinetriphosphate and by stavudine-triphosphate compared with RT from paired wild-type isolates [4]. For stavudine monotherapy, Lin et al. [1], in an analysis on 61 naive and zidovudine-experienced patients on stavudine therapy up to 29 months, described the occurrence of such mutations. Schuurman et al. [5] analysed 16 HIV-1-infected children on stavudine monotherapy whose mothers were antiretroviral-naive during pregnancy. Five patients developed from one to three mutations, conferring resistance to zidovudine (41L, 70R, 210W, 215Y/F), but no multi-drug mutation profiles. The selection of zidovudine-resitance mutations by stavudine was confirmed in the in vitro selection experiments, in which a 215Y mutation was selected in some replicates [5]. For stavudine bitherapy, in clinical trials evaluating the stavudine-didanosine nucleoside combination in treatment-naive patients, TAMs were observed in post-stavudine treatment samples [6 8]. Pellegrin et al. showed that the rate of development of TAMs, at week 24 or 48, in 39 naive patients treated by stavudine plus didanosine, was 36%. At inclusion, there was no statistical difference in HIV-1 load between patients who developed resistance mutations and those who did not. However, the fact that HIV-1 load decrease was higher in patients who maintained a wild-type RT genotype than in those who developed resistance mutations, underlines the importance of evaluating the rates of development of these mutations in different stavudine combination based therapies. Two other clinical trials also found high rates of TAMs in patients treated by stavudine-didanosine combination [6 8]. Stavudine plus lamivudine is one of the most used binucleoside association in antiretroviral combinations. The objective of this study was to assess the genotypic changes in the RT gene in HIV-1, occurring in antiretroviral-naive patients treated by stavudine plus lamivudine. Patients and methods Patients All 44 subjects included in this study were HIV-1 seropositive antiretroviral-naive adults. Thirty-five patients were included in the ALTIS 1 trial, a pilot study that evaluated the use of stavudine plus lamivudine in treatment-naive patients. Nine patients were included in an observational cohort in an academic hospital. All patients were seen at baseline and at 4- weeks intervals thereafter until week 24 or 48. At these times, plasma was collected and stored at 80 C. HIV-1 RNA quantification Quantification of plasma HIV-1 RNA was performed using the Amplicor Monitor assay (Roche Diagnostics, Bazel, Switzerland) with a detection limit of 200 copies. Sequence analysis Plasma RNA taken at week 0, 24 and 48 was used for sequence analysis of the RT gene (codons 1 230). HIV-1 RNA was purified from 1 ml ultracentrifuged ( r.p.m.) plasma using the High Pure Viral RNA purification kit (Boehringer Mannheim, Mannheim, Germany). Plasma HIV-1 RNA was amplified by a one-step RT PCR using the TITAN One Tube Reverse Transcription PCR kit (Boehringer Mannheim), followed by a nested PCR with AmpliTaq Gold (PE Applied Biosystems, Foster City, Calif., USA). The primers Mj3 and Mj4 were used for the one-step RT PCR [9], and primers A35 and NE1-35 were used for the nested PCR [10]. The purified 776 bp DNA fragment obtained was directly sequenced using primers A35 and NE1-35 and the ABI PRISM Dye Terminator Cycle sequencing kit (PE Applied Biosystems. Sequence products were analysed with the ABI PRISM 377 automatic sequencing system, and the sequences were aligned on the HIV-1 consensus B RT gene using the Sequence Navigator software (PE Applied Biosystems). Codons changes that differed from the HIV-1 consensus B RT sequence were considered as mutations. Results RT sequence analysis Genotypes at baseline and at the end of the follow-up are shown in Table 1. At baseline two subjects had TAMs (patient No. 31, T215D; patient No. 34, M41L, L210W, T215Y/S). These particular patterns (215D and 215S) have been described in the context of transmission of zidovudine-resistant viruses [11]. At the end of the follow-up (week 24 or 48), all patients acquired the lamivudine-associated mutation M184V. New International Medical Press

3 HIV-1 RT mutations in d4t/3tc therapy Table 1. Patients with zidovudine/lamivudine combination bitherapy: mutations in HIV-1 reverse transcriptase gene in comparison with the HIV-1 consensus B sequence and plasma viral load (log 10 copies/ml) at baseline and at the end of follow-up RT, reverse transcriptase; WT, wild type Plasma viral load (log 10 copies/ml) RT gene mutations log 10 End-point Patient Baseline (week 24/48 baseline) Baseline [week (W) 24 or week 48] WT M184V (W48) WT M184V (W48) WT M184V (W24) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W24) WT M184V (W24) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W24) WT F116Y, Q151M, M184V (W24) WT M184V (W24) WT M184V (W24) WT M184V (W48) WT M184V (W48) WT M184V (W24) WT M184V (W48) WT M184V (W24) WT M184V (W24) WT M184V (W48) WT M184V (W48) T215D M41l, M184V, T215D (W48) WT M184V (W48) WT M184V (W24) M41L, L210W, T215Y/S M41L, M184V, L210W, T215S (W24) WT M184V (W24) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) WT V75T/A, A98A/G, M184V (W48) WT M184V, T215Y (W48) WT M184V (W48) WT M184V (W48) WT M184V (W48) RT, reverse transcriptase; WT, wild type. Antiviral Therapy 6:3 181

4 M Mouroux et al. TAMs emerged in only two patients: one subject (No. 41) developed a typical TAM (T215Y) and one acquired an additional TAM (patient No. 31: T215D to M41L+T215D). Only one subject (No. 20) developed the particular multidrug-resistance pattern linked to the Q151M mutation (F116Y, Q151M). Only one subject (No. 40) developed a V75T/A mutation. GeneBank accession numbers: AY032669, AY032670, AY032671, AY and AY Plasma viral load Plasma viral loads at baseline (median, 4.86 log 10 copies/ml) and at the end of the follow-up (median, 3.51 log 10 copies/ml) are reported in Table 1. The median drop of viral load was 1.41 log 10 copies/ml for the 44 patients. For the patients who already had TAMs at baseline, the drop in viral loads are close to the median values ( 1.3 log 10 and 1.0 log 10 for patients No. 31 and 34, respectively; Table 1). For patient No. 41 who acquired T215Y mutation, and patient No. 40, who acquired V75T/A mutation, the drop in viral loads ( 2.1 log 10 and 2.5 log 10 copies/ml) was higher than the median value. By contrast, for patient No. 20, who acquired the multi-drug resistant mutations (MDRs), the drop in viral load was lower ( 0.4 log 10 copies/ml; Table 1). Discussion The aim of the present study was to define the HIV-1 RT genotypic resistance patterns that developed in antiretroviral-naive subjects treated with stavudine plus lamivudine dual therapy. The genotypic analyses clearly indicate that this combination administered for weeks selects at a low rate for the emergence TAMs (2.4%) and MDR Q151M mutation profile (2.4%). These results are different from those published previously, particularly for antiretroviralnaive patients treated by stavudine plus didanosine. In these patients, one or more mutations at codons conferring zidovudine resistance were developed by 36%, whereas the Q151M alone or in combination with other thymidine nucleoside analogue resistance or MDR mutations developed in 10% [8]. The discrepancy between results for stavudine/didanosine and stavudine/lamivudine bitherapies could be explained by the development of the M184V mutation in all patients. This hypothesis had already been evoked during the NUCA 3001 trial [12]. In such patients, a low rate of dual resistance to zidovudine/lamivudine was observed (9%) after 1 year of treatment. A mechanism might explain this difference between patients treated by stavudine plus didanosine and those treated by stavudine plus lamivudine or zidovudine plus lamivudine. The lamivudine-associated mutation M184V might have a protective effect against the development of mutations in HIV-1 genome, and in vitro experiments demonstrated that the M184V mutation increased the fidelity of the RT protein. Alternatively, lamivudine-resistant strains of HIV-1 might be attenuated in their replication and therefore may be more susceptible to inhibition by other nucleoside analogues in vivo [13,14]. But this is probably a temporary effect, shown by the genotypic resistance analysis of patients treated for a longer period by stavudine/lamivudine. Among patients receiving stavudine-based regimen, for a median duration of 78 weeks, 38% of patients have selected TAMs [15]. Little is known about the consequences of the development of these TAMs induced by zidovudine or stavudine or a stavudine-based regimen. Several studies have demonstrated that these mutations induced only modest increases of stavudine IC 50 {AUTHOR: IN FULL} but that these increases are correlated with the number of TAMs developed. [5,7,16]. Interpretation remains the residual problem for understanding these data, especially as biochemical tests show decreased stavudine efficiency, even when the IC 50 elevation is not too high [4]. Calvez et al. proposed a re-evaluation of the cutoff of phenotypic resistance to stavudine, considering the data of ALTIS 2 trial, and demonstrated that these weak IC 50 elevations for stavudine have a negative impact on HIV-1 RNA response in patients treated by a combination that includes stavudine [17]. Then, Larder and Wang used a neural network to define the genetic basis of stavudine phenotypic resistance and also provided arguments for a revision of cutoffs previously used for stavudine phenotypes assay interpretation [18]. Another good way to explain the effect of the appearance of TAMs or MDR mutations in antiretroviral-naive patients treated by a stavudine-based regimen, is the evolution of viral load on treatment. In patients treated with stavudine plus didanosine, this phenomenon is associated with a lower virological response and would probably impair subsequent treatments with other nucleoside analogues such as zidovudine [8]. The presence of TAMs at baseline has also been shown to impair the virological response in patients who developed TAMs on zidovudine/zalcitabine combination and switched to stavudine/didanosine combination for 6 months [19] and in those who developed TAMs on zidovudine, didanosine or zalcitabine and switched to stavudine/lamivudine [17]. In our study, for antiretroviral-naive patients treated with stavudine plus lamivudine, evaluation of the impact of TAMs, MDR and V75T mutations selection on the viral load response during the follow-up is difficult because of the International Medical Press

5 HIV-1 RT mutations in d4t/3tc therapy low rate of patients harbouring such mutations at weeks 24 or 28. In conclusion, treatment with stavudine/lamivudine on antiretroviral-naive patients selected a low rate of TAMs and MDR mutations at week 48. One hypothesis explaining these results could be the development of the M184V mutation. Acknowledgments This work was presented in part at: the 3rd International Workshop on HIV Drug Resistance, San Diego, Calif., USA, in June 1999 (abstract 38); the 19th Interdisciplinary Meeting on Anti-Infectious Chemotherapy, Paris, France, in December 1999 (abstract 213,C17); the 4th International Workshop on HIV Drug Resistance, Sitges, Spain, in June 2000 (abstract 54). This study was supported by Agence Nationale de Recherches sur le SIDA (ANRS). We thank Francis Angleraud, Gilles Colin and Michelle Cazabat for technical assistance. References 1. 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